Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or beta (β) cells, appropriate for engraftment. One approach is the generation of functional beta cells from pluripotent stem cells, such as, embryonic stem cells or induced pluripotent cells.
In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, thyroid, thymus, pancreas, gut, and liver will develop from the endoderm via an intermediate stage.
D'Amour et al. described the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (Nature Biotechnology 2005, 23:1534-1541; U.S. Pat. No. 7,704,738). Transplantation of these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of endodermal tissue (U.S. Pat. No. 7,704,738). Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into PDX1 positive cells after addition of FGF-10 and retinoic acid (U.S. Patent App. Pub. No. 2005/0266554). Subsequent transplantation of these pancreatic precursor cells in the fat pad of immune deficient mice resulted in the formation of functional pancreatic endocrine cells following a 3-4 month maturation phase (U.S. Pat. Nos. 7,534,608 and 7,993,920).
Small molecule inhibitors have been used for induction of pancreatic endocrine precursor cells. For example, small molecule inhibitors of TGF-β receptor and BMP receptors (Development 2011, 138:861-871; Diabetes 2011, 60:239-247) have been used to enhance the number of pancreatic endocrine cells. In addition, small molecule activators have also been used to generate definitive endoderm cells or pancreatic endoderm cells (Curr. Opin. Cell Biol. 2009, 21:727-732; Nature Chem. Biol. 2009, 5:258-265).
In general, the process of differentiating progenitor cells to functional beta cells progresses through various stages and strides have been made in improving protocols to generate pancreatic cells from progenitor cells, such as human pluripotent stem cells. Despite these advances in research, each step in the process of differentiating progenitor cells presents a unique challenge. As such, there is still a need for further differentiation protocol development for the purpose of producing functional pancreatic endocrine cells and, in particular, functional beta cells. In particular, it is desirable to provide for in vitro generation of glucose responsive insulin-producing cells capable of the rapid and regulated glucose-stimulated insulin secretion (“GSIS”) observed with functional beta-cells. Specifically, it is desirable to provide a method of producing in vitro functional beta-cells exhibiting an increase in mitochondrial respiration/activity followed by the first phase and second phase of insulin secretion.
GSIS begins by the import of glucose into the beta-cell via a glucose transporter (solute carrier family 2 member 1; SLC2A1; or commonly referred to as glucose transporter 1; GLUT1 for human beta cells), and the metabolism of glucose to pyruvate, through a process named glycolysis. The import of pyruvate into the mitochondria, its metabolism through the TCA (tricarboxylic acid, and subsequent activation of the electron transport chain (“ETC” and referred to here as “mitochondrial activity” or “mitochondrial respiration”) cycle is tightly coupled with insulin exocytosis to ensure the rapid and correct quantity of insulin release.
Functional beta cells within an islet have been shown to secrete insulin upon a sudden increase in glucose concentration in two sequential phases (Henquin et al. Diabetologia (2009) 52(5):739-751). The amplitude and duration of both phases is regulated by the kinetics of the intracellular Ca2+ signal or additive secretion coupling factors. First phase (1st) insulin secretion represents the exocytosis of a small pool of bound and readily releasable insulin granules. The second phase (2nd) of insulin secretion, lower in amplitude but longer in duration, represents the translocation of granules from a reserve pool of granules, and their docking/priming for release. Biphasic GSIS, a key marker of maturation in beta cells, is not detected until the postnatal phase of human development, and is in contrast to the monophasic GSIS seen in immature beta-cells (Otonkoski et al. Diabetes (1988) 37:286-291).
In type 2 diabetics, the 1st phase of GSIS is absent; and the 2nd phase of GSIS is also reduced. Type 1 diabetics, whose beta-cell number is severely reduced via autoimmune attack, are reported to lack a robust biphasic GSIS (Krogvold et al. Diabetes (2015) 64: 2506-2512).